For the past 5 years, we have been trying to identify a substrate(s) of GRAIL, an ubiquitin-protein ligase (E3), which would provide a mechanistic understanding on GRAIL's central role in CD4 T cell unresponsiveness. Our recent data demonstrate that GRAIL is expressed in resting CD4 T cells as well as in anergic CD4 T cells, and our data suggest that GRAIL holds CD4 T cells in cell cycle arrest. Understanding how CD4 T cells are held in cell cycle arrest by GRAIL is the major theme of this proposal. This will be accomplished under 3 specific aims based upon three novel preliminary observations: (1) using the E3 ligase substrate screen we developed under previous funding for this grant, we identified cdc37 as a potential target of GRAIL ubiquitination. Along with upregulation of G1-specific cyclins, cdc37 levels increase as cells enter the cell cycle and undergo proliferation. Studies outlined in this proposal intend to demonstrate that GRAIL expression leads to diminished cdc37 expression and thus to maintenance of cell cycle arrest in CD4 T cells. Additional cell cycle components may be targeted by GRAIL and can be identified using a new E3 ligase substrate screen (global protein stability) developed by our collaborator, Steve Elledge; (2) As we recently demonstrated, naive CD4 T cells can be held in cell cycle arrest by inhibiting either the mTOR or MEK pathways. This inhibition of pS6 blocks the translation of Otub- 1, an epistatic regulator of GRAIL. Our recent data demonstrate that experienced (and possibly memory) CD4 T cells need inhibition through both the MAPK and a non-IL-2R dependent mTOR pAkt pathway in order to block Otub1 translation and resultant GRAIL degradation. We propose to test the hypothesis that antagonists of specific cytokines that bind to their cognate cytokine receptors to block cytokine signaling will provide a cell specific (naive vs. memory CD4 T cell) inhibition of the pAkt pathway and maintain cell cycle arrest; (3) Our data support a model of T cell regulated suppression involving CD4+ CD25+ T regulator cells (Tregs) regulating CD4+ CD25- T effector cells (Teffs) through engagement of CD86 on the Teffs by CTLA-4 expressed on the Tregs. This CTLA-4 induced activity blocks the transcription of il-2 in the Teffs through the induction of icer II by pStat3, and inhibition of IL-2 transcription and translation to maintain GRAIL expression and cell cycle arrest.